G protein-coupled receptor 17 is regulated by WNT pathway during oligodendrocyte precursor cell differentiation.

G protein-coupled receptor 17 (GPR17) and the WNT pathway are critical players of oligodendrocyte (OL) differentiation acting as essential timers in developing brain to achieve fully-myelinating cells. However, whether and how these two systems are related to each other is still unknown. Of interest, both factors are dysregulated in developing and adult brain diseases, including white matter injury and cancer, making the understanding of their reciprocal interactions of potential importance for identifying new targets and strategies for myelin repair. Here, by a combined pharmacological and biotechnological approach, we examined regulatory mechanisms linking WNT signaling to GPR17 expression in OLs. We first analyzed the relative expression of mRNAs encoding for GPR17 and the T cell factor/Lymphoid enhancer-binding factor-1 (TCF/LEF) transcription factors of the canonical WNT/ß-CATENIN pathway, in PDGFRα+ and O4+ OLs during mouse post-natal development. In O4+ cells, Gpr17 mRNA level peaked at post-natal day 14 and then decreased concomitantly to the physiological uprise of WNT tone, as shown by increased Lef1 mRNA level. The link between WNT signaling and GPR17 expression was further reinforced in vitro in primary PDGFRα+ cells and in Oli-neu cells. High WNT tone impaired OL differentiation and drastically reduced GPR17 mRNA and protein levels. In Oli-neu cells, WNT/ß-CATENIN activation repressed Gpr17 promoter activity through both putative WNT response elements (WRE) and upregulation of the inhibitor of DNA-binding protein 2 (Id2). We conclude that the WNT pathway influences OL maturation by repressing GPR17, which could have implications in pathologies characterized by dysregulations of the OL lineage including multiple sclerosis and oligodendroglioma.

Therapeutic potential of stem cells for preterm infant brain damage: Can we move from the heterogeneity of preclinical and clinical studies to established therapeutics?

Acquired perinatal brain injuries are a set of conditions that remains a key challenge for neonatologists and that have significant social, emotional and financial implications for our communities. In our perspective article, we will introduce perinatal brain injury focusing specifically on the events leading to brain damage in preterm born infants and outcomes for these infants. Then we will summarize and discuss the preclinical and clinical studies testing the efficacy of stem cells as neuroprotectants in the last ten years in perinatal brain injury. There are no therapies to treat brain damage in preterm born infants and a primary finding from this review is that there is a scarcity of stem cell trials focused on overcoming brain injuries in these infants. Overall, across all forms of perinatal brain injury there is a remarkable heterogeneity in previous and on-going preclinical and clinical studies in terms of the stem cell type, animal models/patient selection, route and time of administration. Despite the quality of many of the studies this variation makes it difficult to reach a valid consensus for future developments. However, it is clear that stem cells (and stem cell derived exosomes) can reduce perinatal brain injury and our field needs to work collectively to refine an effective protocol for each type of injury. The use of standardized stem cell products and testing these products across multiple models of injury will provide a stronger framework for clinical trials development.

Multicentric experience with interferon gamma therapy in sepsis induced immunosuppression. A case series.

BACKGROUND: The sepsis-induced immunodepression contributes to impaired clinical outcomes of various stress conditions. This syndrome is well documented and characterized by attenuated function of innate and adaptive immune cells. Several pharmacological interventions aimed to restore the immune response are emerging of which interferon-gamma (IFNγ) is one. It is of paramount relevance to obtain clinical information on optimal timing of the IFNγ-treatment, -tolerance, -effectiveness and outcome before performing a RCT. We describe the effects of IFNγ in a cohort of 18 adult and 2 pediatric sepsis patients. METHODS: In this open-label prospective multi-center case-series, IFNγ treatment was initiated in patients selected on clinical and immunological criteria early (< 4 days) or late (> 7 days) following the onset of sepsis. The data collected in 18 adults and 2 liver transplanted pediatric patients were: clinical scores, monocyte expression of HLA-DR (flow cytometry), lymphocyte immune-phenotyping (flow cytometry), IL-6 and IL-10 plasma levels (ELISA), bacterial cultures, disease severity, and mortality. RESULTS: In 15 out of 18 patients IFNγ treatment was associated with an increase of median HLA-DR expression from 2666 [IQ 1547; 4991] to 12,451 [IQ 4166; 19,707], while the absolute number of lymphocyte subpopulations were not affected, except for the decrease number of NK cells 94.5 [23; 136] to 32.5 [13; 90.8] (0.0625)]. Plasma levels of IL-6 464 [201-770] to 108 (89-140) ng/mL (p = 0.04) and IL-10 from IL-10 from 29 [12-59] to 9 [1-15] pg/mL decreased significantly. Three patients who received IFNγ early after ICU admission (<4 days) died. The other patients had a rapid clinical improvement assessed by the SOFA score and bacterial cultures that were repeatedly positive became negative. The 2 pediatric cases improved rapidly, but 1 died for hemorrhagic complication. CONCLUSION: Guided by clinical and immunological monitoring, adjunctive immunotherapy with IFNγ appears well-tolerated in our cases and improves immune host defense in sepsis induced immuno suppression. Randomized clinical studies to assess its potential clinical benefit are warranted.

High plasma level of S100A8/S100A9 and S100A12 at admission indicates a higher risk of death in septic shock patients.

Biomarkers in sepsis for severity, prediction of outcome or reversibility of organ dysfunction are warranted. Measurements of plasma DAMP levels at admission can reflect the severity of cellular damage in septic shock, which might predict the prognosis and reduce the risk of overtreating patients with costly therapies. We measured plasma levels of two DAMPs, S100A8/S100A9 and S100A12 during the first 24 h of admission of septic shock patients. Forty-nine septic shock patients with a similar SOFA scores were selected from our sepsis database to compare a similar proportion of survivors and non-survivors. Plasma levels of S100A8/S100A9 and S100A12 were compared with healthy volunteers using in-house ELISA. Plasma levels of S100A8/S100A9 and S100A12 (5.71 [2.60-13.63] µg/mL and 0.48 [0.22-1.05] µg/mL) were higher in septic shock patients than in healthy volunteers (1.18 [0.74-1.93] µg/mL and 0.09 [0.02-0.39] µg/mL) (P < 0.0001 and P = 0.0030). Levels of S100A8/S100A9 and S100A12 in non-survivors at day 28 (11.70 [2.85-24.36] µg/mL and 0.62 [0.30-1.64] µg/mL) were significantly higher than in survivors (4.59 [2.16-7.47] µg/mL and 0.30 [0.20-0.49] µg/mL) (P = 0.0420 and P = 0.0248) and correlated well (Spearman r = 0.879, P < 0.0001). The high level of plasma calgranulins at admission in septic shock, were higher in non-survivors compared to survivors. These markers could indicate a higher risk of death when SOFA scores are similar and help the stratification of patients for improved care and therapy selection.

Early Sex Differences in the Immune-Inflammatory Responses to Neonatal Ischemic Stroke.

We recently reported that neonatal ischemia induces microglia/macrophage activation three days post-ischemia. We also found that female mice sustained smaller infarcts than males three months post-ischemia. The objective of our current study was to examine whether differential acute neuroinflammatory response and infiltrated immune cells occurs between male and females after three days post-ischemia. Permanent middle cerebral artery occlusion was induced in male and female postnatal 9-day-old (P9) mice, and mice were sacrificed three days after ischemia. Brains were analyzed for mRNA transcription after microglia magnetic cell sorting to evaluate M1 and M2 markers. FACS analysis was performed to assess myeloid infiltration and microglial expression of CX3 chemokine receptor 1 (CX3CR1). Inflammatory cytokine expression and microglia/macrophage activation were analyzed via in situ hybridization combined with immunofluorescence techniques. Lesion volume and cell death were measured. An increase in microglia/macrophages occurred in male versus female mice. The cells exhibited amoeboid morphology, and TNFα and ptgs2 (Cox-2) genes were more expressed in males. More myeloid cell infiltration was found in male versus female brains. However, we did not observe sex-dependent differences in the injured volume or cell death density. Our data show that sex differences in the acute microglial and immune responses to neonatal ischemia are likely both gene- and region-specific.

Downregulation of Blood Monocyte HLA-DR in ICU Patients Is Also Present in Bone Marrow Cells.

BACKGROUND: The downregulation of blood monocyte HLA-DR expression also occurs in tissue infiltrative cells in a context of acute clinical inflammation, especially sepsis. This context favors the development of secondary infections and results from various mechanisms. Little is known about HLA-DR expression on bone marrow (BM) cells of the monocyte lineage, the source of circulating monocytes. This study analyzed the BM HLA-DR expression in ICU patients compared to BM monocytes from non-ICU patients and to blood monocytes of control healthy donors. A potential dysfunction of myeloid differentiation was investigated in a sub-population of these ICU patients to characterize the phenotype of the immature forms of monocytes and granulocytes in BM. METHODS AND FINDINGS: BM and blood were drawn from 33 ICU and 9 non-ICU patients having a BM analysis to precise the etiology of abnormal low count in blood cells. The data were compared with blood cells of 28 control donors. Flow cytometry was used for both HLA-DR expression and phenotyping of immature forms of monocytes and granulocytes. HLA-DR expression was downregulated in both blood and BM monocyte in ICU patients compared to BM of non-ICU patients and blood of control donors. Amplitude of HLA-DR downregulation was comparable in septic and non-septic ICU patients. The phenotype of immature forms of monocytes and granulocytes in BM (n = 11) did not show abnormal myeloid (monocyte + granulocyte) differentiation. CONCLUSION: The downregulation of HLA-DR in BM monocyte lineage is present in ICU patients without major changes in myeloid cells. It may result from a regulation mediated by soluble and/or neuro-endocrine factors present in BM cell microenvironment.

Multicenter testing of the rapid quantification of radical oxygen species in cerebrospinal fluid to diagnose bacterial meningitis.

PURPOSE: Meningitis is a serious concern after traumatic brain injury (TBI) or neurosurgery. This study tested the level of reactive oxygen species (ROS) in cerebrospinal fluid (CSF) to diagnose meningitis in febrile patients several days after trauma or surgery. METHODS: Febrile patients (temperature > 38°C) after TBI or neurosurgery were included prospectively. ROS were measured in CSF within 4 hours after sampling using luminescence in the basal state and after cell stimulation with phorbol 12-myristate 13-acetate (PMA). The study was conducted in a single-center cohort 1 (n = 54, training cohort) and then in a multicenter cohort 2 (n = 136, testing cohort) in the Intensive Care and Neurosurgery departments of two teaching hospitals. The performance of the ROS test was compared with classical CSF criteria, and a diagnostic decision for meningitis was made by two blinded experts. RESULTS: The production of ROS was higher in the CSF of meningitis patients than in non-infected CSF, both in the basal state and after PMA stimulation. In cohort 1, ROS production was associated with a diagnosis of meningitis with an AUC of 0.814 (95% confidence interval (CI) [0.684-0.820]) for steady-state and 0.818 (95% CI [0.655-0.821]) for PMA-activated conditions. The best threshold value obtained in cohort 1 was tested in cohort 2 and showed high negative predictive values and low negative likelihood ratios of 0.94 and 0.36 in the basal state, respectively, and 0.96 and 0.24 after PMA stimulation, respectively. CONCLUSION: The ROS test in CSF appeared suitable for eliminating a diagnosis of bacterial meningitis.

A multicentre study of acute kidney injury in severe sepsis and septic shock: association with inflammatory phenotype and HLA genotype.

BACKGROUND: To investigate the association between severity of acute kidney injury (AKI) and outcome, systemic inflammatory phenotype and HLA genotype in severe sepsis. METHODOLOGY/PRINCIPAL FINDINGS: Prospective multicenter observational study done in 4 intensive care units in two university hospitals. Severe sepsis and septic shock patients with at least 2 organ failures based on the SOFA score were classified: 1) "no AKI", 2) "mild AKI" (grouping stage 1 and 2 of AKIN score) and 3) "severe AKI" (stage 3 of AKIN score). Sequential measurements: The vasopressor dependency index (VDI; dose and types of drugs) to evaluate the association between hemodynamic status and the development of early AKI; plasma levels of IL-10, macrophage migration inhibitory factor (MIF), IL-6 and HLA-DR monocyte expression. Genotyping of the 13 HLA-DRB1 alleles with deduction of presence of HLA-DRB3, -DRB4 and -DRB5 genes. We used multivariate analysis with competitive risk model to study associations. Overall, 176 study patients (146 with septic shock) were classified from AKIN score as "no AKI" (n = 43), "mild AKI" (n = 74) or "severe AKI" (n = 59). The VDI did not differ between groups of AKI. After adjustment, "mild and severe AKI" were an independent risk factor for mortality (HR 2.42 95%CI[1.01-5.83], p = 0.048 and HR 1.99 95%CI[1.30-3.03], p = 0.001 respectively). "Severe AKI" had higher levels of plasma IL-10, MIF and IL-6 compared to "no AKI" and mild AKI (p<0.05 for each), with no difference in mHLA-DR at day 0. HLA-DRB genotyping showed a significantly lower proportion of 4 HLA-DRB alleles among patients requiring renal replacement therapy (RRT) (58%) than in patients with severe AKI who did not receive RRT (84%) (p = 0.004). CONCLUSIONS: AKI severity is independently associated with mortality and plasma IL-10, MIF or IL-6 levels. Presence of 4 alleles of HLA-DRB in severe AKI patients seems associated with a lower need of RRT.

Elevated production of radical oxygen species by polymorphonuclear neutrophils in cerebrospinal fluid infection.

BACKGROUND: Central nervous system infection is a daily concern in neurointensive care; however, diagnosis remains difficult because classical criteria based on cerebrospinal fluid (CSF) analysis are difficult to interpret in post-trauma or neurosurgery patients after recent bleeding. A rapid, specific, sensitive test to diagnose CSF infection would help streamline therapeutic decisions in clinical practice and limit the risk of multiresistant bacteria. We hypothesized that polymorphonuclear neutrophil (PMN) phenotype and radical oxygen species (ROS) production in CSF may be specific to the presence of infection. METHODS: This study included 30 patients with suspected CSF infection with ventricular hemorrhage requiring external ventricular drainage, and 13 patients after trauma or surgery. Criteria for evaluating CSF infection included positive culture and > 100 leukocytes/mm3. Analysis of PMN phenotype was performed using flow cytometry (CD16, CD11b, and CD62L). ROS production was analyzed through luminometry (luminol). RESULTS: Infected CSF exhibited higher production of ROS compared with noninfected CSF. PMNs in CSF exhibited low CD16 and high annexin V expression, suggesting apoptosis. CONCLUSIONS: Measurement of ROS production may discriminate infected from noninfected CSF. This simple test would be easy to employ in clinical practice to improve CSF infection management.

Gene profiling in human blood leucocytes during recovery from septic shock.

OBJECTIVE: To assess blood leucocytes gene profiling during recovery phase of septic shock; to test the relation between encoding gene expression and protein level. STUDY DESIGN: Gene expression levels were studied at days 0, 1, 7 and 28 (D0, 1, 7 and 28) on a dedicated microarray of 340 genes involved in inflammatory processes. SETTINGS: 16-bed intensive care unit, Lariboisière University hospital. PATIENTS: Seventeen septic shock patients enrolled when at least one additional organ dysfunction occurred. MEASUREMENTS AND RESULTS: Changes over time were compared with D0 via the ratio Dx/D0. The time-related gene expression study showed significant changes in ten genes. Among them, S100A8 and S100A12 had a reduced expression over time compared with D0, whereas CD74's expression increased. The microarray results were validated by RT-qPCR for four genes. The S100A8 plasma levels decrease along recovery in parallel with the gene expression decrease. The CD74 gene expression evolution significantly correlated with HLA-DR monocyte expression. CONCLUSIONS: These results are the first description of variations in expression of key inflammatory genes in the course of the septic shock recovery period.

Oxygen consumption of human peripheral blood mononuclear cells in severe human sepsis.

OBJECTIVE: During sepsis, after an initial stimulation immune cells down-regulate their functions, leading to a state of immunosuppression. Because the mechanisms of such down-regulation are unclear, we investigated the hypothesis of an energetic failure of immune cells to participate in immune dysfunction. DESIGN: Cohort of septic shock patients to study peripheral blood mononuclear cells (PBMCs) biological energy in comparison to healthy volunteer cells. SETTING: Critical care unit and laboratory, university hospital. SUBJECTS: Eighteen severe sepsis or septic shock patients and 32 healthy volunteers. INTERVENTIONS: Ex vivo measurement of oxygen consumption in PBMCs taken from patients. The PBMCs' mitochondrial oxidative phosphorylation was investigated using adenosine diphosphate stimulation. The plasma factors implication was tested, using healthy cells incubated in septic plasma, or septic cells incubated in healthy plasma, at different time points of sepsis. The relationship between monocyte human leukocyte antigen-DR expression and bioenergetic results was tested. MEASUREMENTS AND MAIN RESULTS: Baseline oxygen consumption was higher in septic PBMCs (p < .01), with an attenuated response to adenosine diphosphate stimulation (p < .01). Oxygen consumption of healthy PBMCs incubated in septic plasma mimicked the septic cell response, with amplitude depending on the duration of sepsis (days 0-28). Septic cells incubated in healthy plasma partially recovered normal patterns. Septic plasma incubation increased the fraction of decoupling oxygen consumption (p = .021). A relationship between oxygen consumption (baseline or adenosine diphosphate stimulated) and human leukocyte antigen-DR expression was observed for incubation with plasma sampled at different time points of septic shock. CONCLUSION: Energetic failure of PBMCs in sepsis may be a factor associated with the modulation of immune response and human leukocyte antigen-DR phenotype, partially driven by plasma factors.

Levosimendan restores both systolic and diastolic cardiac performance in lipopolysaccharide-treated rabbits: comparison with dobutamine and milrinone.

OBJECTIVE: Current treatment strategies for severe septic conditions (i.e., intravenous fluids, vasopressors, and cardiac inotropes) reestablish fluid balance and improve cardiac systole but do not address diastolic dysfunction. Our study aimed to fully characterize both systolic and diastolic abnormalities of sepsis-associated heart failure and to identify treatment that would support full-cycle cardiac improvement. DESIGN: Endotoxin-injected rabbits, an animal model of abnormal cardiac function in human sepsis, were used to delineate cardiac abnormalities and to examine effects of drug treatments on heart systolic and diastolic function (n = 30); saline-injected animals served as comparators (n = 17). As treatment, three inotropes commonly used for treatment of cardiac failure were infused for 45 mins in separate animal groups-milrinone, dobutamine, and levosimendan. MEASUREMENTS: Variables of left ventricular systolic and diastolic function were assessed with a pressure conductance catheter. Measurements were made before and after endotoxin/saline injection and before and after inotrope treatment. RESULTS: Pressure-volume analyses of the left ventricle showed marked impairment in systolic function and in all indices of diastolic function (isovolumic relaxation time constant, left ventricular end-diastolic pressure, and end-diastolic pressure-volume relationship) in endotoxin-treated rabbits. The inotropes, milrinone, dobutamine, and levosimendan, could each partially or completely restore systolic function in the lipopolysaccharide-treated rabbits. However, only levosimendan therapy led to additional beneficial effects on left ventricular relaxation and diastolic function. CONCLUSIONS: Cardiac failure in severe sepsis results from impairments in both systolic and diastolic functions. Treatment with the calcium sensitizer levosimendan improved both systolic and diastolic cardiac functions in septic animals, but cyclic adenosine monophosphate-dependent inotropes milrinone and dobutamine only improved systolic function.

Mechanisms which mediate the antiapoptotic effects of angiopoietin-1 on endothelial cells.

The main objective of this study was to identify molecular mechanisms through which angiopoietin-1 (Ang-1), a ligand for Tie-2 receptors, influences endothelial cell apoptosis. Human umbilical vein endothelial cells were cultured in a medium enriched with 2% fetal bovine serum (FBS) and growth supplements. Apoptosis was induced over 24 h by reducing FBS to 0.1%. Activation of caspase-9, -8, -7, and -3 and the expression of Bcl-2 family proteins, inhibitors of apoptosis (IAPs), cytochrome c, as well as Smac proteins were evaluated with immunoblotting. Ang-1 clearly attenuated serum deprivation-evoked apoptosis, an effect which required Tie-2 receptor activation. Activation of caspase-9, -7, and -3, but not caspase-8, was inhibited by Ang-1. The inhibitory effects of Ang-1 on apoptosis and caspase activation were reversed by a PI-3 kinase inhibitor (wortmannin). Ang-1 exposure upregulated the expression of Survivin but not XIAP (members of IAPs), reduced the cystosolic levels of Smac, but not that of cytochrome c, and had no effect on the expression of Bcl-2 family proteins. This is the first study to report on the mitochondrial mechanisms through which Ang-1 inhibits apoptosis and to investigate the role of the newly discovered Smac. We conclude that Ang-1 inhibits endothelial cell apoptosis through several pathways, which include PI-3 kinase/AKT activation, inhibition of Smac release from the mitochondria, and upregulation of Survivin protein.